Structural investigations of redox regulation in ATFKBP13 3

Chapter 3 Materials and Methods 3.7.2 Mass spectrometry The precise mass of the protein was determined by MALDI TOF-MS Matrix Assisted Laser Desorption Ionization Time-of-flight Mass Sp

Trang 1

Chapter 3 Materials and Methods

CHAPTER 3 MATERIALS AND METHODS

3.1 EXPRESSIONS AND PURIFICATION OF ATFKBP13

The mature AtFKBP13 gene, which codes 129 amino acids, was cloned into the prokaryotic expression vector pGEX-KG [Rajeev Gupta et al., 2002] The protein was

over expressed in E coli BL21 (DE3) cells The cells were grown in Luria-Bertani

induced with 0.5 mM isopropyl -D-thiogalactopyranoside (IPTG) at 303 K Cell growth was continued at 303 K for 6 h after IPTG induction and cells were harvested by centrifugation at 4,200g (6000 rpm, Beckman JA-8.1000 rotor) for 10 min at 277 K The cell pellet was suspended in ice-cold lysis buffer [20 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 1mM DTT] and homogenized by sonication The crude lysate was centrifuged at

42, 400g (18,000 rpm, Beckman JA-25.50 rotor) for 1 h at 277 K and the cell debris was discarded The supernatant was applied to a GST-affinity column (5 ml glutathione Sepharose 4B) and the contaminant proteins were washed away with wash buffer (lysis buffer plus 400 mM NaCl) and eluted with 50 mM Tris (pH 7.5), 10 mM reduced glutathione 150 units of thrombin were added to the eluate and incubated overnight at

277 K The fusion protein was cleaved efficiently (Fig 3-1) The eluate was dialyzed in

20 mM Tris (pH 7.5), 0.5 M NaCl using 3,500 Da molecular weight cut-off dialysis tubing (Spectra) Thrombin and the cleaved GST were removed by passing through a

Trang 2

5 ml HiTrap benzamidine FF (Pharmacia) column and a 5 ml GSTrap FF column The final purification step was achieved by gel filtration on a HiLoad 16/60 Superdex-75 prep-grade column (Pharmacia) previously equilibrated with a buffer solution containing

50 mM Tris-HCl (pH 7.5) and 150 mM NaCl The purified protein was concentrated to

assay [Bradford, 1976] The protein was then aliquoted as 50 µl per tube, flash frozen with liquid nitrogen and stored at –80 ºC for later use The purified protein was analyzed

on SDS-PAGE and native PAGE The dynamic light-scattering data showed the protein had 70-80% homogeneity as a monomer

Figure 3-1 SDS-Page showing the purification of recombinant

AtFKBP13 using Glutathione matrix M- Marker, Lane 1- Lysate, Lane 2-

M 1 2 3 4 5 6 7

14.3

20.1

29.0

40.0

58.1

Trang 3

Flow-through, Lane 3- Wash, Lane 4- Eluate, Lane 5- Thrombin cleaved,

Lane 6- FPLC purified

3.2 CHLOROPLAST IMPORT ASSAYS AND PROTEIN LOCALIZATION

A radiolabeled AtFKBP13 precursor protein was synthesized by a coupled

[35S]methionine and [35S]cysteine Chloroplasts were isolated from pea and incubated with the precursor proteinas described [Mould and Gray,1998] Import assays containing intact chloroplasts(0.5 mg chlorophyll ml-1), 5 mM methionine, 5 mM cysteine, and10

0.33 M sorbitol] and 45 µlof products from in vitro translation were incubated inlight (100 µmol photons m–2 s–1) for45 min For protein import in the presence of nigericin or

azide for 10 min on ice The 35S-labeledprecursor protein was then added and samples were incubatedat 25 °C for 25 min in light After import, proteinsamples were analyzed

AtFKBP13 (and plastocyanin) in these two fractions

Trang 4

3.3 IN VITRO PROTEIN–PROTEIN INTERACTION ASSAYS

GST-Rieske fusion proteins (1:1 molar ratio), immobilizedon glutathione beads in a final

glutathione, resolved by SDS/PAGE, and detected by westernblot analysis

3.4 PROTEIN EXTRACTION AND WESTERN BLOT ANALYSIS

Total proteins from leaves of 4-week old plants were extractedin a buffer [50 mM

PMSF, 1 mM benzamidine, 5 µg ml-1 leupeptin, 5 µg ml-1 aprotinin, 5 µg ml-1 pepstatin

12,000g for 10 min at 4 °C The supernatantwas collected, and proteins were quantified

chemiluminescence kit (Amersham Pharmacia)

3.5 FKBP13 REDUCTION BY THIOREDOXIN

Reduction experiments were performed using recombinant AtFKBP13 purified

after cleavage by thrombin Reduction by the NADP/thioredoxin system of Escherichia

Trang 5

coli was performed as described [Wong et al., 2003] Recombinant AtFKBP13 (1.5 µg)

was incubated with 0.25 mM NADPH, 0.3 µg NADP-dependent thioredoxin reductase (NTR), and 0.3 µg thioredoxin in 50 mM Tris-HCl (pH 7.5) at 25 °C for 20 min Newly exposed cysteines resulting from disulfide reduction were labeled with the addition of thiol-specific fluorescent probe monobromobimane (mBBr) to 2 mM After labeling, the protein sample was separated by SDS-PAGE [Laemmli, 1970] and the fluorescence recorded using Gel Doc-1000 fitted with a UV 365 nm transilluminator and the Quantity One analysis program (Biorad) Subsequently, the protein pattern was revealed by staining with Coomassie Blue G-250 and captured using a scanner

3.6 PPIASE ASSAY

All assays were carried out using the GST-AtFKBP13 fusion protein GST alone showed no PPIase activity, and the fusion protein showed no reduction of PPIase activity compared to the thrombin-cleaved pure protein PPIase assay was performed using the protocol of Kofron et al (1991), with modifications 45 nM enzyme was incubated with 1.5 mg α-chymotrypsin in reaction buffer [50 mM HEPES (pH 8.0), 100 mM NaCl] and the reaction was allowed to stabilize to 10 °C AtFKBP13 was reduced by incubation

with 0.5 µM of chloroplast m-type or E coli thioredoxin and 500 µM DTT for 20 min at

25 °C The synthetic peptide Suc-Ala-Ala-Pro-Phe-paranitroanilide was dissolved in 470

mM LiCl in trifluoroethanol to maximize the amount of peptide present as the cis-isomer The reaction was started by adding peptide substrate to a final concentration of 60 µM and the catalysis was monitored at 390 nm in a Cary 3E UV/visible spectrometer (Varian)

Trang 6

and data were obtained with the Kinetics application kcat/Km values were calculated as

kobs-k0/[PPIase], where k0 represents the first order rate constant for spontaneous cis-trans isomerisation [Liu et al., 1990]

3.7 BIOPHYSICAL PROPERTIES OF ATFKBP13

3.7.1 Circular dichroism spectroscopy

CD spectroscopy is a monitor of the overall protein secondary structure and is sensitive to conformational changes [Drake, 1994] For CD studies, recombinant AtFKBP13 was prepared, which was eluted as a single peak from the HiLoad 16/60 Superdex-75 gel filtration column The protein were then dialyzed against phosphate buffer of varying pH The secondary structures of the above protein under different pH conditions were examined using their CD spectra Each CD spectrum showed a large negative differential molar extinction coefficient between 210 nm and 220 nm, with a small trough between these wavelengths, as expected for proteins with α-helix and β-sheet contents [Drake, 1994] These characteristics of the native CD spectra are lost at pH 2.5, where the protein is in the substantially unfolded state Fig 3-2 shows the Far-UV spectra of the native state and the unfolded state of this protein The AtFKBP13 at pH 7.0 and at pH 6.0 spectra are very similar, and indicate that they have similarly folded structures They are readily distinguishable from AtFKBP13 at pH 2.5, which shows more of random coil The protein seems to be more stable at pH 4.0 but reveals a reduction of β-strand by 4%

Trang 7

Figure 3-2 CD spectra AtFKBP13 at 20 ºC and varying pH conditions

The differential molar extinction coefficient is shown as a function of

wavelength The sample concentration was 1.5 mg ml-1 Color codes used:

blue (pH 8.5), red (pH 7.0), yellow (pH 6.0), brown (pH 4.0), and green

(pH 2.5)

These results were obtained in three CD sessions with three independent preparations These results suggest that the protein at physiological acidic pH is active and retains its secondary structure

Trang 8

3.7.2 Mass spectrometry

The precise mass of the protein was determined by MALDI TOF-MS (Matrix Assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry) here using a Voyager-DE™ Biospectrometry™ workstation equipped with a 337-nm nitrogen laser

To obtain a good signal-to-noise ratio, 150-200 single shot spectra were collected Saturated sinapinic acid in 50 % acetonitrile was used as the matrix The fractionated protein (0.5 µL) was mixed with 0.5 µL of the matrix and dried on 96 × 2 sample holder prior to the analysis The molecular weight was determined to be 13,527 ± 1.07 Da (Fig 3.3) This showed that the protein was >95% pure

Figure 3-3 Mass Spectrometry for AtFKBP13

Trang 9

3.8 CRYSTALLIZATION AND DATA COLLECTION

3.8.1 Crystallization of AtFKBP13-S2

Initial screening of crystallization conditions followed the sparse-matrix sampling method [Jancarik and Kim, 1991] using Crystal Screen (Hampton Research) and Protein Crystallography Basic Kit (Sigma-Aldrich) Crystallization was performed using the hanging-drop vapor-diffusion method [McPherson, 1990] at 293 K using a 24-well VDX plate (Hampton Research) The size of the droplet, which consisted equal volumes of AtFKBP13-S2 (oxidized AtFKBP13) and reservoir solution, was 5 µl After 3 d, clusters

of twinned plate-like `sheaves' or needle-like crystals were found in three conditions at 2.0 M ammonium sulfate, 5% v/v isopropanol (Crystal Screen II, No 5), 0.1 M HEPES (pH 7.5), 2% PEG 400, 2.0 M ammonium sulfate (Crystal Screen I, No 39) and 0.1 M trisodium citrate (pH 5.6), 20% v/v isopropanol, 20% w/v PEG 4000 (Crystal Screen I,

No 40)

Figure 3-4 AtFKBP13-S2 crystal

Trang 10

These conditions were used as the starting points for optimization experiments using selected reagents from Additive Screens (Hampton Research) and varying the buffer, temperature and protein concentration Single crystals (Fig 3-4) were grown at 293 K in

100 mM Tris (pH 7.9), 8-11% PEG 550 MME, 2.5 M ammonium sulfate Nucleation occurred within 3 days and crystals reached their maximum size in approximately two weeks

3.8.2 Crystallization of AtFKBP13-(SH)2

Crystals of reduced AtFKBP13 [AtFKBP13-(SH)2] were produced in the same way as the oxidized AtFKBP13 crystals The protein was maintained in the reduced form throughout, over a period of two months, by the addition of DTT after crystallization

3.8.3 Data collection and analysis

Prior to data collection, single crystals were rapidly swept through mother liquor containing 20% (v/v) glycerol as a cryoprotectant and were flash-frozen in liquid nitrogen

at 100 K Frozen crystals were screened at 100 K using an in-house X-ray facility (Rigaku RU-H3R rotating-anode X-ray generator operated at 50 kV and 100 mA with an R-AXIS IV imaging-plate detector)

Diffraction data for AtFKBP13-S2 and reduced AtFKBP13 were collected using synchrotron radiation at 100 K (Oxford Cryostream) All diffraction intensities were integrated and scaled using the HKL software package [Otwinowski and Minor, 1997] The crystal data information is given Table 3-1

Trang 11

Table 3-1 Crystal parameters, data-collection and processing statistics

Values in parentheses are for the highest resolution shell, (1.92-1.85) and

(1.95-1.88 Å) respectively

Unit-cell parameters

Data collection

1Rsym = ∑hkl∑i [|I i (hkl) – <I(hkl)>| / I i (hkl)]

Trang 12

3.9.1 Molecular replacement candidates

Sequence alignment with AtFKBP13 as well as spectroscopic studies suggests that FKBPs are structurally homologous After eliminating the NMR structures and the structure of complexes, three possible candidates, human native Fkbp [PDB code: 1D6O,

Burkhard et al., 2000]; yeast Fkbp [PDB code: 1YAT Rotonda et al., 1993]; Bos taurus

Fkbp12 [PDB code: 1FKK, Wilson et al.,1995] were considered as probes for molecular replacement The statistics of each candidate are presented in Table 3-2

Table 3-2 Statistics of Fkbp probe candidates

From the alignment we see that all candidates share high sequence identities with AtFKBP13 The structural homology was also confirmed by making a three-dimensional

Candidate PDB

code

Resolution,

Å

R-value

Sequence identities

Sequence positives

Human native

Fkbp

Bos taurus

Fkbp12

Trang 13

superposition of the given structures using the Dali program [Holm and Sander, 1993] The root mean square deviation (r.m.s.d.) of the Cα-atom positions in all three structures

is within 0.7 Å, confirming that each of these structures can be used as a molecular replacement probe However, yeast FKBP was chosen as the search probe since it has a slightly higher percentage of positive sequence match

3.9.2 Molecular replacement of AtFKBP13

The calculated solvent content of the oxidized AtFKBP13 crystal indicates that 5 AtFKBP13 molecules are present in the asymmetric unit Different combinations of resolution limits, integration radii, and temperature factor distributions were tested for molecular replacement

Table 3-3 Translation function solutions for AtFKBP13-S2 α, β, γ are

Eulerian angles within the AMoRe conventions tx, ty, tz are fractional

translations; R = R-factor

Trang 14

A rotational search using the 20 to 3 Å data in AMoRe [Navaza, 1994 ] resulted

in a set of 10 peaks with correlation coefficients larger than half of that of the first peak The translation search revealed the true nature of these peaks The translation function was performed on the best rotational peaks using the 20 - 3.0 Å resolution range data The height of the produced peaks was limited to half the height of the maximum peak The final output had a correlation coefficient of 30.3% and an R-factor of 54.1% (Table 3-3)

Rigid body fitting of these peaks improved the solution considerably This rigid-body refinement is considered to be another checking procedure, to prove the correctness

of the solutions The least-squares minimization, with respect to the rotational and positional parameters, is performed for each molecule while the others are kept fixed The rigid-body refinement of all the AtFKBP13 molecules yielded the final correlation coefficient of 35.7% and R-factor of 49.2% for 20 to 3 Å data These values confirmed that these solutions were correct

3.9.3 Molecular replacement of AtFKBP13-(SH)2

AtFKBP13-(SH)2 was solved by molecular replacement using the MOLREP program (Vagin and Taplyakov, 1997) and the oxidized AtFKBP13 structure as the probe The Key active residues (Cys5, Cys17, Cys106 and Cys111) were mutated to alanine to prevent any model bias The final model had an R-factor of 35.7% and a correlation value of 63.6%

Trang 15

3.10 STRUCTURE REFINEMENT

The ARP/wARP [Lamzin et al., 2001] software suite was used for automation of model building and refinement using the molecular replacement solution The output of

warpNtrace contained 80 to 98 % polypeptides fragments Main-chain tracing and

building was performed using XtalView [McRee, 1999] The remainder of the structure (cis-prolines, poorly ordered loops and terminal residues for each fragment) was manually completed using the O program [ Jones et al., 1991]

3.10.1 Structure refinement of AtFKBP13

Refinement of AtFKBP13 started with the calculation of 2Fobs-Fcalc and Fobs-Fcalc maps using the molecular replacement solutions The first refinement started from an R-factor of 0.49 with the atomic temperature R-factors fixed set at 20 Å2 In the initial stages, only the atomic coordinates were refined The restraints on geometry were adjusted by adapting the relative weight of the contribution of the X-ray data At the last stages, temperature factors were allowed to refine isotropically and water was picked-up in five cycles of the water-pick up program of CNS

The model was checked for stereochemical correctness using the programs PROCHECK and WHAT_CHECK [Laskowski et al., 1991; Hooft et al., 1996] All stereochemical parameters were flagged as either within normal limits or better when compared to a structure at this resolution There are two regions in the protein with noticeably high temperature factors These include both the N and C termini loop regions

Định dạng
Số trang	17
Dung lượng	242,62 KB